The Glycolytic Switch in Tumors: How Many Players Are Involved?

1. Department of Pathology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, People's Republic of China;2. Guanghua School and Hospital of Stomatology, Sun Yat-sen University, Guangzhou 510055, People's Republic of China;3. State Key Laboratory for Biocontrol, Guangdong Key Laboratory of Pharmaceutical Functional Genes, Key Laboratory of Gene Engineering of the Ministry of Education, Department of Biochemistry, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China.* The two authors contributed equally to this work.

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How
to cite this article:
Yu L, Chen X, Sun X, Wang L, Chen S. The Glycolytic Switch in Tumors: How Many Players Are Involved?. J Cancer 2017; 8(17):3430-3440. doi:10.7150/jca.21125. Available from
http://www.jcancer.org/v08p3430.htm

Abstract

Reprogramming of cellular metabolism is a hallmark of cancers. Cancer cells more readily use glycolysis, an inefficient metabolic pathway for energy metabolism, even when sufficient oxygen is available. This reliance on aerobic glycolysis is called the Warburg effect, and promotes tumorigenesis and malignancy progression. The mechanisms of the glycolytic shift in tumors are not fully understood. Growing evidence demonstrates that many signal molecules, including oncogenes and tumor suppressors, are involved in the process, but how oncogenic signals attenuate mitochondrial function and promote the switch to glycolysis remains unclear. Here, we summarize the current information on several main mediators and discuss their possible mechanisms for triggering the Warburg effect.

Introduction

Reprogramming of glucose metabolism is a key event in tumorigenesis. Cancer cells undergo a metabolic switch from oxidative phosphorylation (OXPHOS) to glycolysis in which a molecule of glucose is degraded to two molecules of pyruvate (Fig 1). Depending on the supply of oxygen for the cells, pyruvate is either reduced to lactate in the absence of oxygen via an anaerobic glycolysis pathway, or oxidized to yield acetyl-coenzyme A in the presence of oxygen and then oxidized completely to CO2 and H2O via citric acid cycle. The majority of cancer cells depend on high rates of glycolysis for growth and survival, even when there is sufficient oxygen [1, 2]. This type of aerobic glycolysis is called the Warburg effect, and the mechanisms underlying this reprogramming are not fully understood. The Warburg effect has long been linked to hypoxia, but it is not solely adaptive to hypoxia, as it also occurs under normoxic conditions [1, 2]. Although mitochondrial dysfunction in cancer cells can cause a shift in energy metabolism, a majority of tumor cells demonstrate normal mitochondrial function and OXPHOS [3-5], and the high glycolytic flux in cancer cells does not mean impairment of OXPHOS [6]. The high rates of glycolysis provide advantages for the survival and growth of cancer cells [7]. Three possible explanations for tumor cell use of the glycolysis pathway, an inefficient metabolic pathway, have been proposed [8, 9]. First, compared to OXPHOS, the rate of ATP production through glycolysis is much more rapid [10]. Secondly, high glycolytic flux provides sufficient glycolytic intermediates to meet the biosynthesis needs of the rapidly proliferating cells [11-13]. Finally, NADPH, derived from the enhanced pentose phosphate pathway (PPP) due to the accumulation of glycolytic intermediates, enables cancer cells to maintain adequate levels of reduced forms of glutathione for resistance to chemotherapeutic agents.

Figure 1

Main steps in glycolysis and possible key enzymes regulated in the Warburg effect. The three reactions catalyzed by hexokinase (HK), phosphofructokinase-1 (PFK1), and pyruvate kinase (PK) in this process are rate-limiting steps. During glycolysis, four molecules of ATP are produced per molecule of oxidized glucose via substrate-level phosphorylation, and the net yield is two molecules of ATP after deduction of two ATPs consumed in phosphorylation. The fate of pyruvate depends largely on the availability of oxygen for the cells. Pyruvate is reduced to lactate under hypoxia via an anaerobic glycolysis pathway or, under aerobic conditions, oxidized to yield acetyl-coenzyme A, which is then oxidized completely to CO2 via the citric acid cycle, resulting in the production of large amounts of ATP. G-6-P, glucose-6-phosphate; G6DP, glucose-6-phosphate dehydrogenase; GLUT, glucose transporter; Fru-2,6-P2, fructose-2,6-bisphosphate; LDH, lactate dehydrogenase; MCT, monocarboxylate transporter; TPI, triose phosphate isomerase.

(Click on the image to enlarge.)

Several mechanisms have been proposed to enable cancer cells to maintain high glycolytic flux [11]. First, phosphofructokinase-1 (PFK1) serves as a critical driver of glycolytic flux. The expression of PFK2 is upregulated in cancer cells and promotes the production of fructose-2,6-bisphospate, which acts as a potent allosteric activator of PFK1 to overcome negative allosteric feedback inhibition of PFK1 by high ATP levels. Second, re-generation of NAD+ and lactate production mediated by lactate dehydrogenase (LDH) is instrumental in maintaining glycolysis. In addition, the expression of pyruvate kinase M2 (PKM2) is upregulated in cancer cells. Allosteric and covalent inhibition of PKM2 channels glycolytic intermediates upstream of pyruvate into biosynthetic pathways [11]. Although metabolic reprogramming has long been observed as a feature of neoplasia and tumor growth, the mechanism triggering and modulating this process remains largely unclear. In this review, we mainly focus on the mechanism underlying the regulation of glycolytic switch in tumors. In addition to signal molecules and transcription factors HIF-1α, c-Myc, Akt, and mTOR, the main regulators which have been well documented, several other regulators including oncogene K-Ras, tumor suppressor p53, energy sensor adenosine monophosphate activated protein kinase (AMPK), non-coding RNAs, and sirtuin family proteins and deacetylation will also be discussed.

Master regulator HIF-1α

Hypoxia-inducible factor-1 (HIF1) consists of two subunits, HIF-1α and HIF-1β, also known as ARNT. Under physiological oxygen levels, the HIF-1α subunit is sensitive to oxygen concentration and is hydroxylated by prolyl-hydroxylases (PHD) and targeted for proteasomal degradation. Increase of reactive oxygen species (ROS) under hypoxia inhibits PHD and stabilizes the HIF-1α subunit. HIF-1α is a master regulator of glycolysis and plays an important role as an activator of aerobic glycolysis and lactate production. It potentiates the transcription of glucose transporters (GLUT) and glycolytic enzymes including GLUT1, hexokinase II (HKII), pyruvate dehydrogenase kinase (PDK), and PKM2 [14-16]. The upregulation of HIF-1-mediated HKII results in a high glycolytic rate in hypoxic solid tumor [17]. Phosphorylation of pyruvate dehydrogenase leads to its inactivation and inhibits conversion of pyruvate into acetyl-CoA for the tricarboxylic acid (TCA) cycle [18, 19]. PKM2 is less catalytically active than is PKM1. A higher level of PKM2 in tumor cells, leading to accumulation of carbohydrate intermediates, facilitates the biosynthesis of macromolecules and tumor cell proliferation. HIF-1α drives expression of many glycolytic enzymes, and hypoxic glycolysis is, in turn, necessary for maintaining HIF-1α activity. This constitutes a novel feed-forward mechanism of glycolysis-HIF-1α signaling (Fig 2) [20].

The mammalian target of rapamycin (mTOR) is also a serine/threonine kinase downstream of Akt and consists of two complexes, mTORC1 and mTORC2. mTOR acts as a central activator of the Warburg effect by inducing expression of glycolytic enzymes under normoxic conditions. mTOR-mediated upregulation of PKM2, a rate-limiting glycolytic enzyme expressed exclusively in tumor cells, is critical to aerobic glycolysis and tumor growth [39]. Tuberous sclerosis protein 1 and 2 complex (TSC1/TSC2) negatively regulates the expression of GLUT3 through the inactivation of mTORC1 signaling [40]. The transmembrane mucin MUC16 increases glycolysis through activation of mTOR. The mTOR-mediated expression of glycolytic proteins involves activation of HIF-1α, NFκB, and c-Myc [39-42]. Upon stimulation, the receptor tyrosine kinases (RTKs) activate membrane PI3K, which recruits and activates Akt. Thus, RTKs-PI3K-Akt-mTOR signaling plays a critical role in the regulation of aerobic glycolysis and tumor growth (Fig 3) [43-45].

Given the high mutation rate of p53 in human tumors, the loss of p53 function could be an important factor contributing to the Warburg effect. It has been determined that tumor-associated mutant p53 (mutp53) drives the Warburg effect under normoxia, and inhibition of glycolysis impairs mutp53-promoting tumorigenesis [66]. Mutant R175H and R273H p53 proteins trigger PKM2 phosphorylation via mTOR signaling [67]. CD147 promotes reprogramming of glucose metabolism by inhibiting the p53-dependent signaling pathway [68].

Non-coding RNAs target glucose transporters and glycolytic enzymes. The downregulation of several miRNAs in some tumors facilitates aerobic glycolysis and promotes the development and progression of the tumors. HIF-1α is a primary target of non-coding RNAs. miRNA absence, or lncRNA-mediated HIF-1α stabilization, enhances HIF-1α activity, contributing to the Warburg effect. PDH, pyruvate dehydrogenase; PDK, pyruvate dehydrogenase kinase.

(Click on the image to enlarge.)

Along with targeting glucose transporters and glycolytic enzymes, many miRNAs exert their functions by targeting HIF-1α, a master regulator of glycolysis (Fig 4). It has been reported that miR-18b [114], miR-186 [115], miR-199a [116, 117], and miR-592 [118] inhibit aerobic glycolysis through directly targeting HIF-1α in several cancer types. Low expression of these miRNAs facilitates aerobic glycolysis and promotes the development and progression of the tumors. The upregulation of HIF-1α under hypoxic conditions, in turn, suppresses miRNA expression and promotes glycolysis [103, 117]. miR-150 targets VHL, a specific E3 ligase for HIF-1α, and promotes the Warburg effect in glioma [119].

Long non-coding RNA (lncRNA) is also an important player in the regulation of the Warburg effect [120, 121]. lncRNA-p21 is hypoxia-responsive and is essential for hypoxia-enhanced glycolysis. It binds to HIF-1α and VHL, disrupting VHL-HIF-1α interaction and VHL-mediated HIF-1α ubiquitination, resulting in HIF-1α accumulation [120]. Long intergenic non-coding RNA for kinase activation (LINK-A), a cytoplasmic lncRNA, mediates BRK-dependent HIF-1α phosphorylation, leading to HIF-1α stabilization under normoxic conditions [121]. LINK-A-dependent normoxic HIF-1α signaling promotes breast cancer glycolysis reprogramming and tumorigenesis [121].

Sirtuin family proteins and deacetylation

Sirtuins are a highly conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases that regulate a large number of cellular processes [122]. Growing evidence demonstrates that sirtuins are involved in the regulation of cancer metabolism [123, 124]. Of the seven mammalian sirtuins (SIRT1-7), SIRT1, SIRT3, and SIRT6 have been implicated in the regulation of glucose utilization [125, 126].

The histone deacetylase SIRT6 has been identified as a tumor suppressor that regulates aerobic glycolysis in cancer cells. Deficiency of SIRT6 in mice results in severe hypoglycemia [127]. SIRT6 acts as a histone H3K9 deacetylase to function as a co-repressor of HIF-1α and Myc and control the expression of multiple glycolytic genes [128, 129]. HIF-1α activity and glycolysis are increased in SIRT6-deficient cells [128, 129]. Lack of SIRT6 can lead to tumor formation even without activation of known oncogenes [129].

SIRT3 is the major deacetylase within the mitochondrial matrix and works as a tumor suppressor by inhibiting the Warburg effect [130, 131]. SIRT3 regulates the stability of HIF-1α via lowering cellular ROS levels [130, 131]. Absence of SIRT3 increases cellular ROS, leading to stabilization of HIF-1α and metabolic reprogramming [131, 132]. In contrast, SIRT3 overexpression represses glycolysis and proliferation in breast cancer cells [131]. The SIRT3-mediated alterations in ROS are attributed to deacetylation and activation of isocitrate dehydrogenase 2 (IDH2) and superoxide dismutase 2 (SOD2) [133]. In addition, SIRT3 deacetylates glutamate oxaloacetate transaminase 2 (GOT2) to inhibit its binding to malate dehydrogenase 2 (MDH2), consequently preventing the malate-aspartate shuttle in the mitochondrial intermembrane space [134]. The malate shuttle is able to restore cytosolic NAD+, which is essential for a high rate of glycolysis. SIRT3 also deacetylates and activates pyruvate dehydrogenase A1 (PDHA1) and PDH phosphatase 1 (PDP1) of the PDH complex (PDC), promoting the conversion of pyruvate to acetyl-CoA for OXPHOS [135, 136]

It has been reported that SIRT1 stimulates the expression of glycolysis genes and the tumor cell proliferation in pancreatic neoplastic lesions [137]. A SIRT1-mTOR/HIF-1α glycolytic pathway is required for differentiation of myeloid-derived suppressor cells into the M1 phenotype [138].

Conclusions

The triggering of the Warburg effect is a complex process with the involvement of multiple regulators (Table 1) [161]. HIF-1α is a master activator. In tumorigenesis, overproduced or mutated growth factors activate transcription factors HIF-1α, NFκB, and c-Myc via the RTKs-PI3K-Akt-mTOR pathway, leading to the expression of glucose transporters and glycolytic enzymes. Oncogene activation and tumor suppressor inactivation during carcinogenesis modify the key signal molecules of the PI3K-Akt-mTOR pathway and downstream HIF-1α activity, promoting glycolytic flux and tumor development. Oncoproteins may also activate sirtuins, a protein deacetylase family, directly suppressing the transcription of glycolytic enzymes or inhibiting HIF-1α and c-Myc expression. Hypoxia and the ROS accumulation and energy depletion resulting from rapid tumor growth further stimulate HIF-1α activity or regulate the production of glycolytic enzymes and glucose transporter through energy sensor AMPK. Absence of miRNAs or lncRNA dysfunction during carcinogenesis promotes aerobic glycolysis via targeting glycolytic enzymes or regulating HIF-1α. A crucial question is whether the Warburg effect is the cause or the effect of cancer. There is no doubt that aerobic glycolysis is a hallmark of tumor metabolism, and is essential to tumor survival and growth. An important focus of study is the stage in tumorigenesis at which reprogramming of glucose metabolism is initiated. Research has revealed that the expression of glycolytic enzymes is modified in the precancerous stage of some tumors [162, 163]. The imaging data also indicated that elevated glycolysis may occur at early-stages of neoplasia and critically contribute to cancer initiation [164, 165]. It has been reported that 14-3-3ζ-mediated upregulation of LDHA in early stage precancerous breast epithelial cells promotes glycolysis, contributing to breast cancer initiation [166]. We have found enhanced expression of several enzymes involved in glycolysis in high grade cervical intraepithelial neoplasia, a typical precancerous lesion of the cervix (Yu et al., unpublished data). This implies that the reprogramming of glucose metabolism occurs at an early stage of carcinogenesis. Additional studies are needed to shed light on this topic.

Increased glycolysis in tumor cells provides a potential target for tumor therapy. Actually, disrupting glycolysis does interfere with tumor growth [167, 168]. Glucose transporters, monocarboxylate transporters, and critical glycolytic enzymes such as HK II, LDHA, PFK, and PKM2 have been proposed as potential targets. Several small molecules including lonidamine, 2-deoxyglucose (2-DG), dichloroacetate, and 3-bromopyruvate (3-BP) have been clinically tested, but many candidates are still under experimental studies [161].

Table 1

The major players in the glycolytic switch and their main features

Regulators

Downstream molecules

Effects

References

Akt

GLUT1, HK II, PDK1, PFK2

+

[32, 33, 35-37]

AMPK

HIF-1α

+, -

[86]

c-Myc

Glucokinase, GLUT1, HKII, LDHA, MCTs, PFK, PK, PKM2

+

[14, 69-75]

HIF-1α

GLUT1, HK II, PDK, PKM2

+

[14-17]

K-Ras

GLUT1

+

[48]

lncRNA

HIF-1α, VHL

+

[120, 121]

miRNAs

Akt, GLUT1, GLUT3, HIF-1α, HKII, LDHA, PDK4, PFK, PFKFB3, PKM2

+, -

[94-105, 107-111]

mTOR

GLUT3, HIF-1α, c-Myc, NFκB, PKM2

+

[39-42]

p53

AMPK, GLUT1, GLUT3, GLUT4, G6PD, miR143, PGM, Pten, RRAD, TIGAR, TSC2

-

[55-60, 62-65]

SIRT1, SIRT3, SIRT6

HIF-1α, Myc, PDHA1, PDP1

-, +

[128-132, 135-137]

Acknowledgements

This study was funded by the National Natural Science Foundation of China (No. 31670788 and No. 81172485), the Ph.D. Program Foundation of Ministry of Education of China (No. 20130171110007), and Open Fund of Guangdong Key Laboratory of Pharmaceutical Functional Genes (No. 2014B030301028).